Anesthetics affect the central nervous system by altering synaptic transmission, but the mechanisms are poorly understood. PSD-95/SAP90 is one of a family of proteins recently shown to physically link synaptic signaling proteins into macromolecular signal transduction structures via PDZ domain interactions. We discovered PSD-95/SAP90 interacts with NMDA receptors and neuronal NOS in the spinal cord. Suppression of PSD-95/SAP90 expression reduced the dose required for inhalational anesthesia. Preliminary data shows clinically relevant concentrations of anesthetics dose-dependently inhibit the PDZ domain-mediated protein interaction between PSD-95 or PSD-93 and the NMDA receptor or neuronal NOS. These inhibitory effects are immediate, potent, reversible, and occur at a hydrophobic peptide-binding groove on the surface of the second PDZ domain of PSD-95. The focus of this proposal is to understand the mechanism by which inhaled anesthetics interact with PDZ domains in neuronal signaling pathways and the biological consequences of these interactions. The aims of the current proposal will: 1) Determine if the effect of inhalational anesthetics on neuronal PDZ domain-mediated protein-protein interactions observed with the NMDA receptor can be generalized to other neuronal signaling pathways using yeast 2-hybrid, co- IP, GST pulldown and plasmon resonance approaches. 2) Define the importance of inhalational anesthetic disruption of PDZ domain-containing protein interactions in the biologic state of anesthesia by investigating the effect of knockout, knockdown and direct inhibition of PSD-93/PSD-95 on MAC of inhalational anesthetics and on locomotor activity, including placing reflex, grasping reflex and righting reflex. Determine the effect of inhalational anesthetic on NMDA mediated neurophysiologic responses in the spinal cord and cortex of mice with and without knockout of PSD-95 or PSD-93. 3) Characterize the biophysical interaction of anesthetics with the PDZ domains. To do so, the effect of mutation of PDZ2 inhalational anesthetic-binding sites and other similar domains on inhalational anesthetic binding to the domains will be evaluated using plasmon resonance, affinity photolabeling, and calorimetry studies.